Endoreduplication in conjunction with tumor progression in an aneuploid laryngeal squamous cell carcinoma

We report the case of a 58-year-old man who presented with a squamous cell carcinoma pT1a G2 of the left vocal cord. Six months after histologically verified complete resection, the patient experienced an endolaryngeal and extralaryngeal local recurrence pT4 pN2b G2. We applied DNA flow cytometry (FCM) and comparative genomic hybridization (CGH) on both primary and recurrent tumor. The primary tumor and the endolaryngeal compartment of the relapse was an aneuploid cell clone with a FCM DNA index of 1.42 and 1.44, respectively. The extralaryngeal compartment showed a shift featuring a DNA index of 2.78. In the primary tumor and in both compartments of the recurrence there was an identical pattern of complex chromosomal imbalances as detected in CGH (CGH karyotype: rev ish enh [8q24.2-q24.3, 10q26.1-q26.3, 11q24-q25, 12q24.2-q23.33,X], dim [4q, 13q14.3-q31], amp[1p36.1-p36.2]). Hence, the recurrence was not associated with further gains and losses of chromosomal material. However, in the anterior part of the recurrence, the aneuploid tumor cell genome had completely doubled, obviously due to endoreduplication. Immunohistochemical analysis of several cell-cycle regulators revealed altered expression of checkpoint proteins, pointing to a complex disturbance in cell-cycle regulation.     

Cell-surface bound pertussis toxin induces polyclonal T cell responses with high levels of interferon-gamma in the absence of interleukin-12

Pertussis toxin (PTx), an exotoxin produced by Bordetella pertussis, has long been used as a mucosal adjuvant. We examined the T cell stimulatory properties of PTx in order to dissect its mechanisms of adjuvanticity. PTx or the B-oligomer of PTx (PTxB) failed to activate purified murine CD4+ or CD8+ T cells, as measured by a lack of proliferation or expression of early T cell activation markers. However, these T cells proliferated extensively in response to the toxin in the presence of syngeneic DC, and proliferation was accompanied by a high level of IFN-gamma production in the absence of IL-12. Interestingly, such responses were independent of signals mediated by MHC-TCR interaction. Both PTx and PTxB were found to bind stably to the surface of DC, and increased the adherence of DC to surrounding cells. These data suggest that polyclonal T cell responses mediated by the toxin are likely to be caused by the toxin bound on the surface of APC, either cross-linking cell surface molecules on T cells, or directly stimulating T cells together with the co-stimulatory molecules expressed on APC. B. pertussis may use this toxin as a mechanism to evade a specific immune response.     

Association of Low Concentrations of Serum Mannose-Binding Protein with Recurrent Infections in Adults

Low concentrations of mannose-binding protein (MBP; also known as mannose-binding lectin) are associated with common opsonic defect in immunodeficient children. We compared the concentrations of MBP in the sera of 47 adults with non-human immunodeficiency virus-related recurrent infections (group I) and 50 healthy adult controls. Mean serum MBP concentrations in the patient group did not differ significantly from those in the control group (P < 0.4). Nevertheless, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in the patient group (21%, P = 0.01) than in the control group (4%). Group II consisted of 73 pediatric and 56 adult patients with recurrent infections. Pediatric patients had significantly lower mean concentrations of serum MBP than their controls (P < 0.005), and there was no significant difference between the concentrations in sera of adult patients and adult controls (P < 0.4). Again, the proportion of individuals with less than 5 ng of serum MBP per ml was significantly larger in both pediatric (22%, P = 0.045) and adult (38%, P = 0.000016) patients than in their respective controls (4%). Our results demonstrate that, as in children, low concentrations of serum MBP can be associated with recurrent infections in adults.     

The psychologist's contribution to primary care: a reappraisal

Developments over the past few years necessitate a reappraisal of the way in which clinical psychologists can best contribute to primary care. Up until now, psychologists have concentrated on those problems which are their concern within psychiatric hospitals. In addition, they have largely adopted a specialist model of service which may not be in the best interests either of primary care or of clinical psychology. If their manner of working is to change, it will be important for psychologists to overcome the developing stereotype of their role. This might involve explicitly negotiating project-orientated commitments which respond to particular priorities in different settings.     

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.     

Spontaneous Cytokine Production and Its Effect on Induced Production

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Multiparameter flow cytometry is often used to examine cell-specific cytokine production after in vitro phorbol 12-myristate 13-acetate and ionomycin induction, with brefeldin A or other agents added to inhibit protein secretion. Spontaneous ex vivo production reportedly rarely occurs. We examined the spontaneous production of interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) by peripheral-blood B lymphocytes, T cells, CD8⁻ T cells, CD8⁺ T cells, CD3⁻ CD16/56⁺ lymphocytes (natural killer [NK] cells), CD3⁺ CD16/56⁺ lymphocytes (natural T [NT] cells), and/or monocytes of 316 acutely ill hospitalized persons and 62 healthy adults in Malawi, Africa. We also evaluated the relationship between spontaneous and induced cytokine production. In patients, spontaneous TNF-α production occurred most frequently, followed in descending order by IFN-γ, IL-8, IL-4, IL-10, IL-6, and IL-2. Various cells of 60 patients spontaneously produced TNF-α; for 12 of these patients, TNF-α was the only cytokine produced spontaneously. Spontaneous cytokine production was most frequent in the immunoregulatory cells, NK and NT. For IL-2, IL-4, IL-6, IL-8, and IL-10, spontaneous cytokine production was associated with greater induced production. For TNF-α and IFN-γ, the relationships varied by cell type. For healthy adults, IL-6 was the cytokine most often produced spontaneously. Spontaneous cytokine production was not unusual in these acutely ill and healthy persons living in an area where human immunodeficiency virus, mycobacterial, malaria, and assorted parasitic infections are endemic. In such populations, spontaneous, as well as induced, cell-specific cytokine production should be measured and evaluated in relation to various disease states.     

Filarial Nematode Parasites Secrete a Homologue of the Human Cytokine Macrophage Migration Inhibitory Factor

Filarial nematode parasites establish long-term chronic infections in the context of an antiparasite immunity that is strongly biased toward a Th2 response. The mechanisms that lead to this Th2 bias toward filarial antigens are not clear, but one possibility is that the parasites produce molecules that have the capacity to proactively modify their immunological environment. Here we report that filarial parasites of humans secrete a homologue of the human proinflammatory cytokine macrophage migration inhibitory factor (MIF) that has the capability of modifying the activity of human monocytes/macrophages. A cDNA clone isolated from a Brugia malayi infective-stage larva expression library encoded a 12.5-kDa protein product (Bm-MIF) with 42% identity to human and murine MIF. MIF homologues were also found to be expressed in the related filarial species Wuchereria bancrofti and Onchocerca volvulus. Bm-mif was transcribed by adult and larval parasites, and the protein product was found in somatic extracts and in the parasite’s excretory-secretory products. Immunohistocytochemistry revealed that Bm-MIF was localized to cells of the hypodermis/lateral chord, the uterine wall, and larvae developing in utero. Unexpectedly, the activities of recombinant Bm-MIF and human MIF on human monocytes/macrophages were found to be similar. When placed with monocytes/macrophages in a cell migration assay, Bm-MIF inhibited random migration. When placed away from cells, Bm-MIF induced an increase in monocyte/macrophage migration that was specifically inhibited by neutralizing anti-Bm-MIF antibodies. Bm-MIF is the first demonstration that helminth parasites produce cytokine homologues that have the potential to modify host immune responses to promote parasite survival.     

The morphology and mechanical properties of endomysium in series-fibred muscles: variations with muscle length

Summary In the series-fibred muscle architecture commonly found in large muscles of mammals and birds, the intrafasciculary-terminating muscle fibres have no direct tendinous attachments. Contractile force produced in these fibres must be transmitted between adjacent muscle fibres via the endomysial connective tissue which separates them. The endomysium is thus an essential mechanical component in such muscles. Studies of motor end-plate banding patterns and the frequent occurrence of tapering ends of fibres within the fascicles of the bovine sternomandibularis muscle show it to be a series-fibred muscle. Sodium hydroxide digestion of fixed samples of this muscle to remove the myofibrillar apparatus revealed the endomysium to be a disordered planar network of mainly curvilinear collagen fibrils.The orientation distribution of the collagen fibrils in the endomysial network was measured by image analysis of scanning electron micrographs. Analysis of endomysial preparations from muscle fixed at sarcomere lengths between 1–4 m showed that the orientation distribution of collagen fibrils is quantitatively related to muscle length. At rest sarcomere length the collagen fibril network is not completely random, but has a slight circumferential bias. The orientation distribution shows a progressive shift towards the circumferential direction at short sarcomere lengths and towards the longitudinal direction at long sarcomere lengths. The relationship between the number-weighted mean collagen orientation and sarcomere length was compared to two geometric models of network behaviour, the isoareal and constant shape models. both fitted the data reasonably, although the constant shape model described the rate of change of mean orientation more closely.From fibrous composites theory, the reinforcement efficiency factor, was calculated from the measured collagen fibril orientation distributions. These calculations predict a non-linearly increasing longitudinal tensile modulus for the endomysium with increasing sarcomere length, in agreement with its known non-linear properties, but confirm that the tensile properties of the endomysium are unsuitable for transmission of tensile force from muscle fibres contracting near rest length. This reinforces a previous interpretation that contractile force is transmitted between neighbouring muscle fibres by trans-laminar shear through the endomysium rather than by in-plane tension.     

Characterization of a monoclonal anti-Bw4 antibody (Tü109): Evidence for similar epitopes on the Bw4 and Bw6 antigens

The production and serologic, as well as immunochemical properties of a cytotoxic murine IgG monoclonal antibody (Tü109) that precipitates HLA-class I molecules, are described. In the microcytotoxicity assay Tü109 supernatant was demonstrated on a panel of 424 HLA-ABC, -DR, -DQ, -MT typed normal Caucasian blood donors to define an epitope on HLA-B locus molecules in great association with the supertypic specificity Bw4. Reactivity of supernatant showed MHC linked inheritance of the Tü109 determinant and discriminated the HLA-Bw4/Bw6 associated HLA-B locus split antigens. Weak or lack of binding on lymphocytes from some HLA-Bw4 heterozygous individuals, particularly typing for HLA-Bw44, appeared to be due to qualitative and/or quantitative variations of HLA-B locus molecules on the cell surface. With Tü109 ascites fluid, however, extra-reactivity on all HLA-Bw6⁺ cells was demonstrated. Preferential binding of supernatant to HLA-Bw4, but reactivity of ascites fluid with HLA-Bw6⁺ molecules in addition, was furthermore confirmed by IEF analysis of antigens immunoprecipitated with Tü109 from cell lysates. Thus the antibody may help to analyze the evolutionary relationship of the diallelic specificities Bw4 and Bw6.